USING THE SYNTHETIC APPROACH TO IMPROVING IMMUNOGENICITY OF VACCINES Synthetic Vaccines against Shigella dysenteriae Type 1 and Shigella sonnei Shigella dysenteriae type 1 and Shigella sonnei are Gram-negative human pathogens that cause endemic and epidemic dysentery throughout the world. Despite their discovery many years ago, there are still no licensed vaccines against these pathogens, which have developed resistance to most available antibiotics. Our approach to vaccine development against these bacteria is based on the demonstration that serum antibodies against the O-specific polysaccharides (O-SPs) of Gram-negative bacteria are important for host protection. While O-SPs are nonimmunogenic, presumably because of their low molecular weight, covalent conjugates of an immunogenic protein and the O-SP of S. dysenteriae type 1 and S. sonnei have been shown to elicit significant anti-O-SP antibody levels of the IgG isotype, which may be boosted by repeated injections. We surmised that an improved vaccine might be constructed from chemically defined oligosaccharide fragments of O-SPs, which are devoid of biological contamination, carry a uniform molecular weight, and can be characterized by physicochemical methods. In earlier studies we have found that oligosaccharides as small as an octasaccharide can generate substantial amounts of O-SP specific antibodies in mice when coupled to a protein, even if administered without an adjuvant. Based on this finding, most of our efforts in the past year have been directed towards the chemical synthesis of octa-, dodeca-, and hexadecasaccharides under clean-laboratory conditions in sufficient quantities. Our earlier synthetic experience served as the basis of this work. However, numerous chemical problems were encountered during the scale-up that necessitated further synthetic explorations. As a result, we were able to assemble fully protected oligosaccharides up to a hexadecasaccharide and have started the deprotection steps. We anticipate that the spacer-linked target oligosaccharides will be available before the end of this year. In order to determine the role of the non-reducing terminal monosaccharide in eliciting O-SP specific antibodies, we have prepared oligosaccharides in the range of hexa- to tridecamers in forms suitable for covalent coupling to a protein. As a further exploration into synthesizing saccharide-protein constructs of enhanced anti-carbohydrate immunogenicity, we continued our work towards "linearly arranged clusters" of an octasaccharide fragment of the O-SP in which the octasaccharide fragments of the O-SP corresponding to two contiguous repeating units are interconnected by extended linkers at predefined sites. These saccharides will be coupled to a protein and the saccharide-protein constructs so obtained will be tested for their immunogenicity in mice. In an effort to improve the efficiency of the covalent attachment of the synthetic oligosaccharides to proteins, we have developed a new linker containing an activated carbon-carbon double bond that is likely to improve the predictability and efficiency of our conjugation method based on the Diels-Alder cycloaddition reaction. Synthetic Vaccines against Group A Neisseria meningitidis Group A Neisseria meningitidis causes endemic and epidemic meningococcal meningitis worldwide. Although a vaccine containing the purified capsular polysaccharide (CPS) of this bacterium has been available for years, it is not sufficiently immunogenic in infants, who are at the highest risk. Our program to develop a more efficient vaccine is based on the assumption that protein conjugates of relatively short fragments of the CPS (i. e. that can be made available by controlled chemical synthesis) of Group A N. meningitidis will be more immunogenic in infants than the currently available vaccine. The CPS of Group A N. meningitidis consists of alpha-linked N-acetyl-mannosaminyl residues that are interconnected by an anomerically located phosphodiester linkage. The native polysaccharide is nonstoichiometrically O-acetylated at the O-3 position. We have already synthesized oligomeric fragments of the CPS that lacks the O-acetyl group at O-3 and demonstrated that those constructs were recognized by a polyclonal antibody pool raised against formalin-killed Group A Neisseria meningitides. As an extension of this project, we have designed synthetic schemes to prepare analogs of the CPS that contain the O-acetyl group at O-3. We have already assembled a monomer of the repeating unit that will be used in chemical synthesis towards extended fragments suitable for bioconjugation to proteins.